Rat pheochromocytoma PC12 cells and bovine adrenal chromaffin cells are used to study the mechanism of secretion and its regulation by Ca2+ and GTP-binding proteins. Most of our work is done with digitonin-permeabilized cells. The release of catecholamines from these permeabilized cells is both Ca2+ and ATP dependent. Protein phosphatases and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of secretion. Our results suggest that norepinephrine secretion by both PC12 cells and chromaffin cells is regulated by a Ca2+-stimulated phosphorylation. Digitonin-permeabilized chromaffin cells are used to identify proteins involved in the secretory response. Incubation of these permeabilized cells results in a loss of Ca2+-dependent secretion. The addition of cytosolic proteins restores this secretory activity. We have fractionated cytosolic extracts and demonstrated that more than one protein is required for Ca2+-dependent secretion. We have purified one of these proteins and are in the process of characterizing it. Also in contrast to a published report, we have shown that calpactin is not involved in this restoration. Secretion of norepinephrine by digitonin-permeabilized PCI2 cells can also be stimulated by the addition of GTPYS. We are continuing to try to determine the mechanism by which GTPYS induces secretion in these cells. Several aspects of this stimulation suggest that the protein responsible for this stimulation might be a low molecular GTP-binding protein rather than a heterotrimeric GTP-binding protein.